Detection of the presence of specific biomolecules, such as DNA or RNA sequences, proteins, antigens, antibodies, etc., in a sample is required for a variety of experimental, diagnostic and therapeutic purposes. A multitude of assays are available for detecting proteinaceous biomolecules such as gel electrophoretogram, HPLC, affinity chromatography, as well as other assays which are performed by use of an appropriately labelled probe. While such assays are satisfactory where the proteinaceous biomolecule to be detected is present in sufficiently large quantities, they are at times not sensitive enough to allow detection of minute quantities of biomolecules.
DNA or RNA sequences can be detected by the use of a labelled probe. Where the DNA or RNA sequences to be detected present in only very small amounts, they have to be amplified by methods such as LCR (ligase chain reaction), SSR (self-sustained sequence replication) or PCR (polymerase chain-reaction).
Although amplification methods such as PCR have had an extremely high impact on basic research, they have been slow in making the transition to the clinical setting. The primary reason for this is that the requirement for automation combined with the clinical environment of the samples, have yielded processes that are complex, slow and expensive. The need for protein enzymes with their high sensitivity to environmental factors necessitates a very controlled environment in which they are to operate. Typically, a clinical sample contains many components that can interfere with the enzyme's ability to perform its catalytic activity. In addition, the standard methods that are used for sample preparation to release the nucleic acids, such as Guanidine thiocyanate or Phenol extraction are unsuitable for protein based enzymatic activity and it is therefore necessary to remove the target nucleic acid from sample preparation.
Ribozymes are typically RNA molecules having enzyme-like catalytic activities that are usually of cleavage, splicing or ligation of nucleic acid sequences. The known substrates for ribozymes are RNA molecules although there have been some indications that ribozymes may act on DNA molecules and on proteins.
Natural ribozymes which participate in intracellular reaction work in cis, catalyzing only a single turnover, and are usually self-modified during the reaction. However, ribozymes can be engineered to act in trans, in a truly catalytic manner, with a turnover greater than one and without being self-modified. Two distinct regions can be identified in a ribozyme: the binding region which gives the ribozyme its specificity through hybridization to a specific nucleic acid sequence (and possibly also to specific proteins), and a catalytic region which gives the ribozyme the activity of cleavage, ligation or splicing. Each class of ribozymes cleaves a different sequence of nucleotides using a distinct mechanism of action. Each class is further distinguished by the number of nucleotide bases that are essential for its catalytic activity and by the degree of the specificity of the ribozyme and the target sequence (Robert H. Simons, Annual Review of Biochenistry, 61, pp. 641-671, (1992)).
It has recently been proposed to use ribozymes in order to treat diseases or genetic disorders by cleaving a target RNA, such as viral RNA or messenger RNA transcribed from genes that should be turned off. This method is proposed as an alternative to blockage of the RNA transcript by the use of antisense sequences. Owing to the catalytic nature of the ribozyme, a single ribozyme molecule cleaves many molecules of target RNA and therefore therapeutical activity is achieved in relatively lower concentrations than those required in an antisense treatment (WO 96/23569).
The use of ribozymes for diagnostic purposes has been only seldomly mentioned. WO 94/13833 describes a method for detecting nucleic acid molecules in a solution by tailoring a specific ribozyme molecule having two regions, one complementary to the nucleic acid sequence to be detected, and the other complementary to a co-target molecule bearing a detectable label. The ribozyme is able to specifically and reversibly bind both a selected target nucleic acid sequence and to the labelled co-target. When both the target and the co-target are bound, the ribozyme undergoes a conformational change which renders it active and able to cleave the label off the co-target, and the free label can then be detected. Upon cleavage of the co-target, the ribozyme is able to re-associate with an additional co-target, cleaving more label and producing more detectable signals.
Although the inventors of WO 94/13833 termed their invention "amplification of signal" there is actually no amplification in the number of ribozymes produced, but rather the reaction is purely an enzymatic reaction, wherein the catalytic substance (in this case the ribozyme) cleaves the substrate (in this case the co-target) and then disassociate and cleave another substrate. There is no true amplification of the number of active ribozymes involved in the reaction occurs.